Solution structure of the recombinant human oncoprotein p13 MTCP1

The human oncoprotein p13 ^MTCP1 is coded by the MTCP1 gene, a gene involved in chromosomal translocations associated with T-cell prolymphocytic leukemia, a rare form of human leukemia with a mature T-cell phenotype. The primary sequence of p13 ^MTCP1 is highly and only homologous to that of p14 ^TCL1 , a product coded by the gene TCL1 which is also involved in T-cell prolymphocytic leukemia. These two proteins probably represent the first members of a new family of oncogenic proteins. We present the three-dimensional solution structure of the recombinant p13 ^MTCP1 determined by homonuclear proton two-dimensional NMR methods at 600 MHz. After proton resonance assignments, a total of 1253 distance restraints and 64 dihedral restraints were collected. The solution structure of p13 ^MTCP1 is presented as a set of 20 DYANA structures. The rmsd values with respect to the mean structure for the backbone and all heavy atoms for the conformer family are 1.07 ± 0.19 and 1.71 ± 0.17 Å, when the structured core of the protein (residues 11–103) is considered. The solution structure of p13 ^MTCP1 consists of an orthogonal -barrel, composed of eight antiparallel -strands which present an original arrangement. The two -pleated loops which emerge from this barrel might constitute the interaction surface with a potential molecular partner.     

Structural basis for the co-activation of protein kinase B by T-cell leukemia-1 (TCL1) family proto-oncoproteins

Chromosomal translocations leading to overexpression of p14(TCL1) and its homologue p13(MTCP1) are hallmarks of several human T-cell malignancies (1). p14(TCL1)/p13(MTCP1) co-activate protein kinase B (PKB, also named Akt) by binding to its pleckstrin homology (PH) domain, suggesting that p14(TCL1)/p13(MTCP1) induce T-cell leukemia by promoting anti-apoptotic signals via PKB (2, 3). Here we combined fluorescence anisotropy, NMR, and small angle x-ray-scattering measurements to determine the affinities, molecular interfaces, and low resolution structure of the complex formed between PKBbeta-PH and p14(TCL1)/p13(MTCP1). We show that p14(TCL1)/p13(MTCP1) target PKB-PH at a site that has not yet been observed in PH-protein interactions. Located opposite the phospholipid binding pocket and distal from known protein-protein interaction sites on PH domains, the binding of dimeric TCL1 proteins to this site would allow the crosslinking of two PKB molecules at the cellular membrane in a preactivated conformation without disrupting certain PH-ligand interactions. Thus this interaction could serve to strengthen membrane association, promote trans-phosphorylation, hinder deactivation of PKB, and involve PKB in a multi-protein complex, explaining the array of known effects of TCL1. The binding sites on both proteins present attractive drug targets against leukemia caused by TCL1 proteins.     

Refined solution structure and backbone dynamics of 15N-labeled C12A-p8MTCP studied by NMR relaxation

MTCP1 (for Mature-T-Cell Proliferation) was the first gene unequivocally identified in the group of uncommon leukemias with a mature phenotype. The three-dimensional solution structure of the human p8^MTCP protein encoded by the MTCP1 oncogene has been previously determined by homonuclear proton two-dimensional NMR methods at 600 MHz: it consists of an original scaffold comprising three -helices, associated with a new cysteine motif. Two of the helices are covalently paired by two disulfide bridges, forming an -hairpin which resembles an antiparallel coiled-coil. The third helix is orientated roughly parallel to the plane defined by the -antiparallel motif and appears less well defined. In order to gain more insight into the details of this new scaffold, we uniformly labeled with nitrogen-15 a mutant of this protein (C12A-p8^MTCP1) in which the unbound cysteine at position 12 has been replaced by an alanine residue, thus allowing reproducibly high yields of recombinant protein. The refined structure benefits from 211 additional NOEs, extracted from ¹⁵N-edited 3D experiments, and from a nearly complete set of angular restraints allowing the estimation of the helical content of the structured part of the protein. Moreover, measurements of ¹⁵ N spin relaxation times and heteronuclear ¹⁵ N¹HNOEs provided additional insights into the dynamics of the protein backbone. The analysis of the linear correlation between J(0) and J() was used to interpret relaxation parameters. It appears that the apparent relative disorder seen in helix III is not simply due to a lack of experimental constraints, but associated with substantial contributions of sub-nanosecond motions in this segment.     

Observation of intersubunit NOEs in a dimeric P22 Mnt repressor mutant by a time-shared [15N, 13C] double half-filter technique

Summary A time-shared [¹⁵N, ¹³C] half-filter technique is presented, which can be used to study proton-proton NOEs between biomolecules. The filter is demonstrated in a 2D [¹⁵N, ¹³C] double filtered NOESY experiment of a dimeric Mnt repressor mutant consisting of completely [¹⁵N, ¹³C] labeled monomer and unlabeled monomer. The benefit of this combined [¹⁵N, ¹³C] half-filter is that a single NMR experiment can be designed that yields all NOE interactions between labeled and unlabeled protons ((¹³C, ¹⁴N/¹²C), (¹⁵N, ¹⁴N/¹²C), (¹²C, ¹⁵N/¹³C) and (¹⁴N, ¹⁵N/¹³C)) in the protein, where conventional half-filters would require at least three separate NMR experiments to obtain these NOEs. The intermonomer NOEs of the Mnt mutant confirmed the secondary structure of the DNA-binding domain as an antiparallel ribbon, formed from an N-terminal segment contributed by each monomer. Moreover, several intersubunit NOEs were characterized in the C-terminal part of the Mnt mutant for which no structural data is available yet.     

A 3D NOESY-(HCACO)NH experiment for the measurement of NOEs involving 1Hα in 13C-/15N-labeled proteins dissolved in H2O

Summary A 3D NOESY-(HCACO)NH experiment is described that transfers NOEs from ¹H to the backbone ¹H^N in the succeeding residue for detection. Using this strategy, NOEs involving ¹H protons that resonate exactly at the water frequency can be detected. NOEs from an overlapping ¹H proton that is attached to degenerate ¹³C can also be resolved. The performance of this approach is demonstrated for the ¹³C-/¹⁵N-labeled Hck/SH2 dissolved in H₂O.     

Structure refinement of flexible proteins using dipolar couplings: Application to the protein p8MTCP1

The present study deals with the relevance of using mobility-averaged dipolar couplings for the structure refinement of flexible proteins. The 68-residue protein p8^MTCP1 has been chosen as model for this study. Its solution state consists mainly of three -helices. The two N-terminal helices are strapped in a well-determined -hairpin, whereas, due to an intrinsic mobility, the position of the third helix is less well defined in the NMR structure. To further characterize the degrees of freedom of this helix, we have measured the dipolar coupling constants in the backbone of p8^MTCP1 in a bicellar medium. We show here that including D _HN ^dip dipolar couplings in the structure calculation protocol improves the structure of the -hairpin but not the positioning of the third helix. This is due to the motional averaging of the dipolar couplings measured in the last helix. Performing two calculations with different force constants for the dipolar restraints highlights the inconstancy of these mobility-averaged dipolar couplings. Alternatively, prior to any structure calculations, comparing the values of the dipolar couplings measured in helix III to values back-calculated from an ideal helix demonstrates that they are atypical for a helix. This can be partly attributed to mobility effects since the inclusion of the ¹⁵N relaxation derived order parameter allows for a better fit.     

Chemical shift assignments and secondary structure of the Grb2 SH2 domain by heteronuclear NMR spectroscopy

Summary The growth factor receptor-bound protein-2 (Grb2) is an adaptor protein that mediates signal transduction pathways. Chemical shift assignments were obtained for the SH2 domain of Grb2 by heteronuclear NMR spectroscopy, employing the uniformly ¹³C-/¹⁵N-enriched protein as well as the protein containing selectively ¹⁵N-enriched amino acids. Using the Chemical Shift Index (CSI) method, the chemical shift indices of four nuclei, ¹H, ¹³C, ¹³C and ¹³CO, were used to derive the secondary structure of the protein. Nuclear Overhauser enhancements (NOEs) were then employed to confirm the secondary structure. The CSI results were compared to the secondary structural elements predicted for the Grb2 SH2 domain from a sequence alignment [Lee et al. (1994) Structure, 2, 423–438]. The core structure of the SH2 domain contains an antiparallel -sheet and two -helices. In general, the secondary structural elements determined from the CSI method agree well with those predicted from the sequence alignment.Abbreviations crk viral p47^gag-crk - EGF epidermal growth factor - GAP GTPase-activating protein - PI3K phosphatidylinositol-3-kinase - PLC- phospholipase-C-, shc, src homologous and collagen - src sarcoma family of nonreceptor tyrosine kinase     

Sequential1H-NMR assignments of neurotoxin III from the sea anemoneHeteractis macrodactylus and structural comparison with related toxins

The complete sequence-specific assignment of resonances in the¹H-NMR spectrum of the polypeptide neurotoxin III (Hm III) from the sea anemoneHeteractis macrodactylus is described. Comparison of the chemical shifts and pattern of NOEs for Hm III with those for the related toxin Hp III fromHeteractis paumotensis, which differs only in the substitution of Asn for Tyr at position 11, shows that the overall secondary and tertiary structures are conserved. The largest differences in chemical shift caused by the substitution at position 11 are observed for the NH resonances of Arg-13, Thr-14, Ala-15, Leu-17, and Cys-26. The CH resonances influenced most are those of ASP-6, Gly-9, Leu-17, and Glu-42, while the most affected CH resonances are from Leu-17, Glu-28, and Lys-32. The absence of long-range NOEs to the aromatic ring of Tyr-11 as well as the lack of significant chemical shift effects on residues outside the loop comprising residues 7–16 confirm that this part of the loop makes no long-lived contacts with the rest of the molecule. The deviations from random coil shifts of Hm III are compared with those of the related anemone toxins Hp II, Hp III, and toxin I fromStichodactyla helianthus (Sh I). The similarity in deviations in chemical shift as a function of sequence position for these four toxins emphasizes the overall structural homology among these polypeptides.     

An investigation into the solution structure of the single-stranded DNA undecamer 5′d AAGTGTGATAT by means of nuclear Overhauser enhancement measurements

A 500-MHz ¹H-NMR study on the single-stranded DNA undecamer (11-mer) 5d AAGTGTGATAT is presented. Using a combination of one-dimensional pre-steady-state nuclear Overhauser enhancement (NOE) measurements and two-dimensional homonuclear J-correlated spectroscopy, virtually complete resonance assignments are obtained. The relative magnitudes of the intra- and internucleotide NOEs indicate that the overall structure of the single-stranded 11-mer is a right-handed B-type helix with extensive base stacking. Within this overall structure there is quite a large degree of variability, as exemplified by variations in glycosidic bond and sugar pucker conformations, most likely determined by base sequence.Abbreviations NOE nuclear Overhauser effect - COSY twodimensional homonuclear J-correlated spectroscopy - 11-mer undecamer - EDTA sodium ethylenediamine tetraacetate - HPLC nigh-pressure liquid chromatography - DSS 4,4-dimethylsilapentane-1-sulfonate     

Purification and characterization of recombinant forms of murine Tcl1 proteins

The TCL1 gene, which is located on chromosome 14, plays a major role in human hematopoietic malignancies and encodes a 14-kDa protein whose function has not been determined. This gene is expressed in pre-B cells, in immature thymocytes, and, at low levels, in activated T cells but not in peripheral mature B cells and in normal cells. The Tcl1 protein is similar in its primary structure to a protein encoded by the mature T-cell proliferation gene (MTCP1). The MTCP1 gene is located on the X chromosome and has been shown to be involved in rare chromosomal translocations in T-cell proliferative diseases. The murine TCL1 gene resides on mouse chromosome 12 and is homologous to the human TCL1 and MTCP1 genes. Murine Tcl1 protein has 116 amino acid residues and shares 50% sequence identity with human Tcl1, while the human and mouse Mtcp1 are nearly identical, with conservative differences in only six residues. The TCL1 and MTCP1 genes appear to be members of a family of genes involved in lymphoid proliferation and T-cell malignancies. Our laboratory has undertaken the study of the Tcl1 and Mtcp1 proteins to determine the structure and the function of these related proteins. In the present report, we have produced, using a bacterial expression system, the purified murine Tcl1 protein and a mutant form of murine Tcl1 protein containing a cysteine to alanine mutation at amino acid position 85. The recombinant proteins were purified by chromatography on a Ni-NTA resin followed by reverse-phase FPLC using a buffer system at pH 7.9 and a polymer-based reverse-phase column. The murine Tcl1 recombinant protein displays limited solubility and forms disulfide-linked dimers and oligomers, while the mutant murine Tcl1 C86A protein has increased solubility and does not form higher order oligomers. The purified recombinant murine proteins were characterized by N-terminal sequence analysis, mass spectrometry, and circular dichroism spectroscopy. Initial results indicate that the mutant murine Tcl1 C86A protein is suitable for both NMR and X-ray crystallographic methods of structure determination.     

1H and 15N resonance assignments and secondary structure of cellular retinoic acid-binding protein with and without bound ligand

Summary Sequence-specific assignments for the ¹H and ¹⁵N backbone resonances of cellular retinoic acid-binding protein (CRABP), with and without the bound ligand, have been obtained. Most of the side-chain resonances of both apo- and holo-CRABP have also been assigned. The assignments have been obtained using two-dimensional homonuclear and heteronuclear NMR data, and three-dimensional ¹H-¹⁵N TOCSY-HMQC and NOESY-HMQC experiments. The secondary structure, deduced from nuclear Overhauser effects, amide H/D exchange rates and H chemical shifts, is analogous in both forms of the protein and is completely consistent with a model of CRABP that had been constructed by homology with the crystal structure of myelin P2 protein [Zhang et al. (1992) Protein Struct. Funct. Genet., 13, 87–99]. This model comprises two five-stranded -sheets that form a sandwich or -clam structure, and a short N-terminal helix-turn-helix motif that closes the binding cavity between the two sheets. Comparison of the data obtained for apo- and holo-CRABP indicates that a region around the C-terminus of the second helix is much more flexible in the apo-protein. Our data provide experimental evidence for the hypothesis that the ligand-binding mechanism of CRABP, and of other homologous proteins that bind hydrophobic ligands in the cytoplasm, involves opening of a portal to allow entry of the ligand into the cavity.     

Stereospecific assignment of β-methylene protons in larger proteins using 3D15N-separated Hartmann-Hahn and13C-separated rotating frame Overhauser spectroscopy

Summary ³J _x coupling constants and complementary nuclear Overhauser data on the intraresidue C ^x H–CH distances form an essential part of the data needed to obtain stereospecific assignments of -methylene protons in proteins. In this paper we show that information regarding the magnitude of the³J _x coupling constants can be extracted from a semi-quantitative interpretation of relative peak intensities in a 3D¹⁵N-separated¹H–¹H Hartmann-Hahn¹H–¹⁵N multiple quantum coherence (HOHAHA-HMQC) spectrum. In addition, we demonstrate that reliable information on the intraresidue C ^x H–CH distances, free of systematic errors arising from spin diffusion, can be obtained from a 3D¹³C-separated¹H–¹H rotating frame Overhauser effect¹H–¹³C multiple quantum coherence (ROESY-HMQC) spectrum. The applicability of these experiments to larger proteins is illustrated with respect to interleukin-1, a protein of 153 residues and 17.4 kDa molecular weight.Abbreviations 1L-1 interleukin-1 - NOE nuclear Overhauser effect - ROE rotating frame Overhauser effect - HOHAHA homonuclear Hartmann-Hahn spectroscopy - NOESY nuclear Overhauser enhancement spectroscopy - ROESY rotating frame Overhauser spectroscopy - HMQC heteronuclear multiple quantum coherence spectroscopy     

3D Triple-resonance NMR techniques for the sequential assignment of NH and 15N resonances in 15N- and 13C-labelled proteins

Summary Two new 3D ¹H-¹⁵N-¹³C triple-resonance experiments are presented which provide sequential cross peaks between the amide proton of one residue and the amide nitrogen of the preceding and succeeding residues or the amide proton of one residue and the amide proton of the preceding and succeeding residues, respectively. These experiments, which we term 3D-HN(CA)NNH and 3D-H(NCA)NNH, utilize an optimized magnetization transfer via the ²J_NC coupling to establish the sequential assignment of backbone NH and ¹⁵N resonances. In contrast to NH-NH connectivities observable in homonuclear NOESY spectra, the assignments from the 3D-H(NCA)NNH experiment are conformation independent to a first-order approximation. Thus the assignments obtained from these experiments can be used as either confirmation of assignments obtained from a conventional homonuclear approach or as an initial step in the analysis of backbone resonances according to Ikura et al. (1990) [Biochemistry, 29, 4659–4667]. Both techniques were applied to uniformly ¹⁵N- and ¹³C-labelled ribonuclease T1.     

Rapid corepressor exchange from the trp-repressor/operator complex: An NMR study of [ul-13C/15N]-l-tryptophan

Summary [ul-¹³C/¹⁵N]-l-tryptophan was prepared biosynthetically and its dynamic properties and intermolecular interaction with a complex of Escherichia coli trp-repressor and a 20 base-pair operator DNA were studied by heteronuclear isotope-edited NMR experiments. The resonances of the free and bound corepressor (l-Trp) were unambiguously identified from gradient-enhanced ¹⁵N–¹H HSQC, ¹³C–¹H HSQC, ¹³C-and ¹⁵N-edited 2D NOESY spectra. The exchange off-rate of the corepressor between the bound and free states was determined to be 3.4±0.52 s^–1 at 45°C, almost three orders of magnitude faster than the dissociation of the protein-DNA complex. Examination of the experimental NOE buildup curves indicates that it may be desirable to use longer mixing times than would normally be used for a large molecule, in order to detect weak intermolecular NOEs in the presence of exchange. Intermolecular NOEs from bound corepressor to trp-repressor and DNA were analyzed with respect to the mechanism of ligand exchange. This analysis suggests that, in order for the ligand to diffuse out of the complex, there must be significant movement or breathing of the protein and/or DNA.Abbreviations NOESY nuclear Overhauser enhancement spectroscopy - HSQC heteronuclear single-quantum coherence - PFG pulsed field gradient - l-Trp l-tryptophan     

Precise vicinal coupling constants3JHNα in proteins from nonlinear fits of J-modulated [15N,1H]-COSY experiments

Summary Improved experimental schemes for the recently introduced J-modulated [¹⁵N,¹H]-correlation experiment for measurements of the homonuclear amide proton-C proton vicinal coupling constants.³J_HN, in uniformly¹⁵N-labeled proteins are described, and a nonlinear fit procedure is presented for quantitative evaluation of³J_HN. The method was first tested with the N-terminal DNA-binding domain of the 434 repressor (M=7.3 kDa), where at 13 C precise values of³J_HN in the range 2.0–9.5 Hz were obtained for all residues with resolved¹⁵N-¹H cross peaks. It was then applied to theAntennapedia homeodomain complexed to a synthetic 14-base pair DNA fragment (molecular weight of the complex 18 kDa). The³J_HN values measured were found to be in excellent agreement with those predicted from the secondary structure of this protein in the complex.Abbreviations and symbols NOE nuclear Overhauser effect - COSY two-dimensional correlated spectroscopy - ³J_HN or J homonuclear vicinal amide proton-C proton coupling constant - 434 repressor(1–69) N-terminal DNA-binding domain of the 434 repressor comprising 69 residues     

Differential regulation of Akt kinase isoforms by the members of the TCL1 oncogene family.

The members of the TCL1 proto-oncogene family (TCL1, MTCP1, and TCL1b) bind to Akt1, increasing its phosphorylation status and kinase activity. This is thought to be secondary to the formation of TCL1-Akt oligomers within which Akt is preferentially phosphorylated. Here we show that, in contrast to Akt1 and Akt2, which bind to all members of the TCL1 family, Akt3 specifically interacts with TCL1 but not with MTCP1 or TCL1b. This association is functional, as the presence of TCL1 but not MTCP1 or TCL1b increased Akt3 kinase activity in in vitro kinase assays. Functional specificity is determined by the Akt pleckstrin homology domain as chimeric Akt1, where Akt1 PH domain was replaced by that of Akt3 was no longer able to interact with MTCP1 or TCL1b and its kinase activity was solely enhanced by TCL1. Moreover, we show that, in TCL1-overexpressing SUPT-11 T-cell leukemia and P3HR-1 Burkitt's lymphoma cell lines, TCL1 interacts with endogenous Akt1, Akt2, and Akt3. TCL1 enhanced hetero-oligomerization of Akt1 with Akt3 and as a consequence facilitated transphosphorylation of Akt molecules, which may contribute to Akt activation and TCL1-induced leukemogenesis in vivo.     

Sequential Main-Chain Proton and Carbon Resonance Assignments for Wild-Type Yeast Iso-1 Ferrocytochrome c and Identification of Secondary Structural Elements

Yeast iso-1 cytochrome c is a naturally occurring protein that possesses an unusually reactive Cysl02 that imbues iso-1 with a complicated solution chemistry which includes spontaneous dimerization and poorly characterized redox reactions. For this reason previous studies of this typical member of the c-type cytochromes have been relegated to variant proteins in which the 102 position has been mutated, with most common changes involving serine and threonine. However, we have determined sequential ¹H resonance assignments for the wild-type native protein because it is the actual participant in yeast mitochondrial electron transfer processes and because the wild-type native protein should be the fundamental assignment basis. In addition to ¹H resonance assignments for 97 of 106 amino acids, we have also provided an extensive database of long-range NOEs. Comparison of these NOEs and a chemical shift index based upon -H resonances has lead to identification of solution secondary structural elements that are consistent with the solid-state crystal structure. Although there is currently no efficient expression system that would facilitate isotope labeling of iso-1 cytochrome c, we tried to assess the usefulness of future heteronuclear experiments by using natural-abundance ¹H/¹³C HMQC experiments to unambiguously assign 35 -C resonances.     

1H and 15N resonance assignments and solution secondary structure of oxidized Desulfovibrio vulgaris flavodoxin determined by heteronuclear three-dimensional NMR spectroscopy

Summary Sequence-specific ¹H and ¹⁵N resonance assignments have been made for all 145 non-prolyl residues and for the flavin cofactor in oxidized Desulfovibrio vulgaris flavodoxin. Assignments were obtained by recording and analyzing ¹H–¹⁵N heteronuclear three-dimensional NMR experiments on uniformly ¹⁵N-enriched protein, pH 6.5, at 300 K. Many of the side-chain resonances have also been assigned. Observed medium-and long-range NOEs, in combination with ³J_NH coupling constants and ¹H_N exchange data, indicate that the secondary structure consists of a five-stranded parallel -sheet and four -helices, with a topology identical to that determined previously by X-ray crystallographic methods. One helix, which is distorted in the X-ray structure, is non-regular in solution as well. Several protein-flavin NOEs, which serve to dock the flavin ligand to its binding site, have also been identified. Based on fast-exchange into ²H₂O, the ¹H^N3 proton of the isoalloxazine ring is solvent accessible and not strongly hydrogen-bonded in the flavin binding site, in contrast to what has been observed in several other flavodoxins. The resonance assignments presented here can form the basis for assigning single-site mutant flavodoxins and for correlating structural differences between wild-type and mutant flavodoxins with altered redox potentials.     

NMR structure determination of the tetramerization domain of the Mnt repressor: An asymmetric α-helical assembly in slow exchange

The structure and dynamics of the chymotryptic tetramerization domain of the Mnt repressor of Salmonella bacteriophage P22 have been studied by NMR spectroscopy. Two sets of resonances (A and B) were found, representing the asymmetry within the homotetramer. Triple-resonance techniques were used to obtain unambiguous assignments of the A and B resonances. Intra-monomeric NOEs, which were distinguished from the inter-monomeric NOEs by exploiting ¹³C/¹⁵N-filtered NOE experiments, demonstrated a continuous -helix of approximately seven turns for both the A and B monomers. The asymmetry facilitated the interpretation of inter-subunit NOEs, whereas the antiparallel alignment of the subunits allowed further discrimination of inter-monomeric NOEs. The three-dimensional structure revealed an unusual asymmetric packing of a dimer of two antiparallel right-handed intertwined coiled -helices. The A and B forms exchange on a timescale of seconds by a mechanism that probably involves a relative sliding of the two coiled coils. The amide proton solvent exchange rates demonstrate a stable tetrameric structure. The essential role of Tyr 78 in oligomerization of Mnt, found by previous mutagenesis studies, can be explained by the many hydrophobic and hydrogen bonding interactions that this residue participates in with adjacent monomers.     

Heteronuclear three-dimensional NMR spectroscopy of a partially denatured protein: The A-state of human ubiquitin

Summary Human ubiquitin is a 76-residue protein that serves as a protein degradation signal when conjugated to another protein. Ubiquitin has been shown to exist in at least three states: native (N-state), unfolded (U-state), and, when dissolved in 60% methanol:40% water at pH 2.0, partially folded (A-state). If the A-state represents an intermediate in the folding pathway of ubiquitin, comparison of the known structure of the N-state with that of the A-state may lead to an understanding of the folding pathway. Insights into the structural basis for ubiquitin's role in protein degradation may also be obtained. To this end we determined the secondary structure of the A-state using heteronuclear three-dimensional NMR spectroscopy of uniformly ¹⁵N-enriched ubiquitin. Sequence-specific ¹H and ¹⁵N resonance assignments were made for more than 90% of the residues in the A-state. The assignments were made by concerted analysis of three-dimensional ¹H-¹⁵N NOESY-HMQC and TOCSY-HMQC data sets. Because of ¹H chemical shift degeneracies, the increased resolution provided by the ¹⁵N dimension was critical. Analysis of short- and long-range NOEs indicated that only the first two strands of -sheet, comprising residues 2–17, remain in the A-state, compared to five strands in the N-state. NOEs indicative of an -helix, comprising residues 25–33, were also identified. These residues were also helical in the N-state. In the N-state, residues in this helix were in contact with residues from the first two strands of -sheet. It is likely, therefore, that residues 1–33 comprise a folded domain in the A-state of ubiquitin. On the basis of ¹H chemical shifts and weak short-range NOEs, residues 34–76 do not adopt a rigid secondary structure but favor a helical conformation. This observation may be related to the helix-inducing effects of the methanol present. The secondary structure presented here differs from and is more thorough than that determined previously by two-dimensional ¹H methods [Harding et al. (1991) Biochemistry, 30, 3120–3128].